PURPOSE: The purpose of this study was to elucidate in vitro the effect of elevated glucose on glucose uptake in the cells comprising the inner and outer blood-retinal barriers: human retinal pigment epithelial (hRPE) and human retinal vascular endothelial (hRVE) cells. METHODS: Primary cultures of hRPE and hRVE cells grown in 5.5 or 22 mM glucose or in 22 mM mannitol were used to measure the rates of glucose uptake with [(3)H]-3-O-methyl glucose as a tracer. GLUT1 expression was measured by Northern and western blot analyses. Cellular fractionation was performed by differential centrifugation. GLUT1 overexpression was accomplished by adenoviral transduction. RESULTS: Increasing media glucose from 5.5 to 22 mM for 30 minutes caused a 1.9-fold increase in the V(max) of glucose uptake in hRPE cells and a 2.5-fold increase in hRVE cells. These increases were nonosmotic and glucose specific, in that the exposure to 22 mM mannitol did not affect the V(max) of glucose uptake. mRNA, total protein expression, and translocation of GLUT1, the glucose transporter predominantly expressed in hRPE and hRVE cells, were not affected by 22 mM glucose for up to 48 hours. High-glucose effects on V(max) were abolished with 10 microg/mL of the microtubule assembly inhibitor nocodazole. hRPE cells transduced to overexpress GLUT1 showed a 1.5-fold increase in the V(max) for glucose uptake versus control-transduced cells. However, the magnitude of glucose-induced increase in glucose uptake was the same in GLUT1- and control-transduced cells. CONCLUSIONS: High glucose induced 1.9- and 2.5-fold increases in the V(max) of glucose uptake in hRPE and hRVE cells, respectively. These increases were not due to an increase in GLUT1 expression. The increases were dependent on microtubule integrity, but not on GLUT1 translocation. The mechanism of the increases is unknown. GLUT1 regulating protein(s) and/or novel glucose transporter(s) may be involved in the regulation of glucose uptake by glucose in the cells that comprise the blood-retinal barrier.
PURPOSE: The purpose of this study was to elucidate in vitro the effect of elevated glucose on glucose uptake in the cells comprising the inner and outer blood-retinal barriers: humanretinal pigment epithelial (hRPE) and human retinal vascular endothelial (hRVE) cells. METHODS: Primary cultures of hRPE and hRVE cells grown in 5.5 or 22 mM glucose or in 22 mM mannitol were used to measure the rates of glucose uptake with [(3)H]-3-O-methyl glucose as a tracer. GLUT1 expression was measured by Northern and western blot analyses. Cellular fractionation was performed by differential centrifugation. GLUT1 overexpression was accomplished by adenoviral transduction. RESULTS: Increasing media glucose from 5.5 to 22 mM for 30 minutes caused a 1.9-fold increase in the V(max) of glucose uptake in hRPE cells and a 2.5-fold increase in hRVE cells. These increases were nonosmotic and glucose specific, in that the exposure to 22 mM mannitol did not affect the V(max) of glucose uptake. mRNA, total protein expression, and translocation of GLUT1, the glucose transporter predominantly expressed in hRPE and hRVE cells, were not affected by 22 mM glucose for up to 48 hours. High-glucose effects on V(max) were abolished with 10 microg/mL of the microtubule assembly inhibitor nocodazole. hRPE cells transduced to overexpress GLUT1 showed a 1.5-fold increase in the V(max) for glucose uptake versus control-transduced cells. However, the magnitude of glucose-induced increase in glucose uptake was the same in GLUT1- and control-transduced cells. CONCLUSIONS: High glucose induced 1.9- and 2.5-fold increases in the V(max) of glucose uptake in hRPE and hRVE cells, respectively. These increases were not due to an increase in GLUT1 expression. The increases were dependent on microtubule integrity, but not on GLUT1 translocation. The mechanism of the increases is unknown. GLUT1 regulating protein(s) and/or novel glucose transporter(s) may be involved in the regulation of glucose uptake by glucose in the cells that comprise the blood-retinal barrier.
Authors: Veedamali S Subramanian; Zainab M Mohammed; Andres Molina; Jonathan S Marchant; Nosratola D Vaziri; Hamid M Said Journal: J Physiol Date: 2007-04-26 Impact factor: 5.182